JP2006514034A - Stimulates hematopoietic function by in vitro activated immune cells - Google Patents

Abstract

Experimental protocol for restoring bone marrow histology and / or blood cell counts to normal levels in patients with aplastic anemia by activating human blood cells and feeding to patients. The experimental protocol involves culturing blood cells in the presence of cytokines and ionophores. One thing to emphasize is that this summary is submitted according to certain rules. According to this rule, the summary of the disclosed technology must be able to be found by the searcher or other user immediately after reading the summary. The summary should not be used to understand or limit the scope or meaning of claim 37 C.F.R..sctn. 1.72 (b).

Description

  The present invention relates to a method for treating aplastic anemia, anemia and thrombocytopenic purpura, and more particularly to a method for treating aplastic anemia, anemia and thrombocytopenic purpura using activated blood cells cultured in vitro. Furthermore, the invention relates to a method for activating cells and a corresponding method for cell culture.

  Aplastic anemia is a disease caused by reduced hematopoietic function. Abnormalities of varying degrees occur in the regenerative function of all blood cells in the patient. In many cases, the etiology of this disease cannot be elucidated, but radiation, benzine compounds, viruses (such as hepatitis virus), environmental toxins, over-the-counter drugs and prescription drugs can damage the bone marrow and eventually cause programmed cell death of bone marrow stem cells ( leading to apoptosis) seems to be the cause of this disease. However, regardless of the etiology, all patients have similar clinical symptoms and progression. Aplastic anemia is a disease that occurs frequently in both young and old, regardless of gender. Every year, about 2 to 6 out of a million people in the world get this disease, of which there are more Orientals than Europeans and Americans. Pathogenic factors for this disease generally include innate causes, pregnancy, viruses, and drugs and chemicals.

  Of the pathogenic factors for aplastic anemia, the best known is exposure to drugs and chemicals. Pathogenic factors such as chloramphenicol, benzine, ionizing radiation, and anticancer drugs cause poor regeneration depending on the amount used or exposed, but the severity varies from person to person. Return to. However, in the case of pathogenic agents such as insecticides, some antispasmodics, and antibiotics, reactions that are not related to the amount used can occur, so that the response cannot be predicted through blood tests at the time of administration. Sometimes regeneration failure occurs after the administration is stopped. Unlike patients with congenital aplastic anemia, patients with aplastic anemia caused by drugs and toxins have approximately the same clinical characteristics, demographic characteristics and prognosis, and approximately the same response to treatment Indicates.

  In mild cases, benzine-induced regenerative anemia disappears if exposure to benzine is eliminated, but there is still no effective and safe treatment if severe or continuous blood transfusion is required. At present, there is nothing other than bone marrow transplantation.

  In general, for patients with mild regenerative anemia, the principle is to treat as little as possible, eliminate pathogenic factors, and aim for natural healing. However, for patients with young severe regenerative anemia, the only treatment is to transplant bone marrow from an HLA-matched donor. Bone marrow transplant has a cure rate of about 80%, but if the donor and patient loci do not match, the survival rate drops to 10-20%. Patients who have undergone bone marrow transplantation also have complications such as graft rejection, acute or chronic graft-versus-host disease, infection and other organ-specific damage, as well as long-term solid cancer. The risk of becoming is quite high.

  The inventor of the present invention studied the treatment of anemia such as anemia, aplastic anemia, and thrombocytopenic purpura using cultured (activated) blood cells. In one embodiment of the invention, anemia is treated by injecting a patient with an effective amount of blood cells. The blood cells used may be cultured under conditions where cytokines and ionophores are present. Cytokines and ionophores may have effective concentrations. Cytokines may contain interleukin-2 and macrophage colony stimulating factor. The ionophore may contain A23187.

  In another embodiment of the present invention, a method for treating anemia by administering in vitro cultured blood cells to a patient has been proposed. The blood cells used may be of a therapeutically effective amount. The blood cells used may be the patient's own, the same type as the patient's own blood cells, or those that can be accepted from the point of view of immunity. Further, the blood cells used may be cultured under conditions where cytokines and ionophores having an effective amount are present.

  In another embodiment of the present invention, a method for culturing blood cells in the presence of cytokines and ionophores was proposed. By way of example, the blood cells are cultured under conditions in which effective amounts of cytokines and ionophores are present, cultured in a medium with or without mammalian serum, or 2 to 200 hours or It may be cultured for a longer time, or may be cultured at 30 to 42 ° C. or other temperature conditions. The cytokine may contain interleukin-2 and / or granulocyte-macrophage colony stimulating factor. The ionophore may contain A23187.

  The present invention relates to a method for treating anemia such as anemia, aplastic anemia and thrombocytopenic purpura by administering a therapeutically effective amount of in vitro cultured blood cells to a patient. The above-mentioned “therapeutically effective amount” means that anemia patients are provided with a sufficient amount of the activated blood cells to significantly increase the number of blood cells, in other words, red blood cells, white blood cells, The concentration of factors and cells (natural components of blood) made by the patient's bone marrow, such as platelets, will be very low. The blood cells used for the culture may be those of the patient himself or those from a donor who can accept them from the viewpoint of immunity. One method of in vitro culture is to first take a blood sample (eg, 10-100 ml) from a patient or an acceptable donor from the angle of immunity, then isolate the blood sample, obtain blood cells and culture . The above “donor that can be accepted from the angle of immunity” means that the tissue containing the donor's blood cells does not have a medical response to the receptor (eg hemolytic anemia, heart failure, renal failure, etc.). It means not to wake up. The blood cells are separated from serum by a method such as centrifugation. The separated blood cells are cultured under sterile conditions using a medium containing cytokine (or cell stimulating factor) or ionophore, or both. The culture time and culture temperature of the separated blood cells are, for example, within 1 hour, 1 hour or more, and in other examples, 10 to 200 hours, 20 to 80 hours, or 30 to 60 hours are increased. For example, 30-42 ° C., 32-40 ° C., or 37-38 ° C., but all times and temperatures within these clearly specified ranges are the times and temperatures claimed by this disclosure. I think that this is common sense in the patent field, so I will not make a luxury here.

  Anemia can be treated by the above methods. In general, anemia is a disease caused by low levels of blood components (cells, etc.) that are the product of bone marrow in the blood. Anemia, aplastic anemia, thrombocytopenic purpura (Thrombocytopenic purpura). Anemia may be considered a lack of blood components, but in some cases is limited to a lack of red blood cells. Aplastic anemia is a lack of peripheral blood components. Thrombocytopenic purpura (for example, congenital thrombocytopenic purpura) means that the number of platelets is small. In the following, the treatment of aplastic anemia will be described in detail with examples, but of course this treatment can be applied to other anemias.

The cultured activated blood cells were washed (eg, washed twice with sterile saline) and a therapeutically effective amount was given to the patient. As a method of administration, intravenous injection is recommended as one of the good methods. Although it may be administered once, it may be divided into several times during a certain period. For example, the activated blood cells may be administered once a week for a total of 4 weeks, or twice a week, every 10 days, every 14 days, every 21 days, or at other intervals and periods. good. In addition, the administration interval may be changed during treatment. For example, the initial administration interval of the blood cells is set once a day, twice a week, once a week, or once every two weeks. The dose for each treatment may be, for example, 1 × 10 ⁵ to 2 × 10 ⁸ depending on the age and condition of the patient. The treatment period (when the activated blood cells are administered) depends on the supply of activated blood cells and the patient's reaction. A patient's reaction can be judged, for example, when the number of blood cells has returned to a normal level, the histological structure of the bone marrow has become normal, or the condition of the whole body has improved. Needless to say, blood samples can be taken from the patient during or after the initial treatment, and activated blood cells can be cultured for later treatment. Furthermore, activated blood cells used for the initial treatment or all treatments may be cultured in blood samples from donors that can be accepted from an immunization angle. In addition, activated blood cells cultured in a blood sample from a donor that can be accepted from the angle of immunity at the first stage of treatment are administered, and then the patient's own blood sample is taken, and the activated blood cells are cultured in it. May be administered to the patient.

  According to the latest definition, severe aplastic anemia is pancytopenia that satisfies two or more of the following three conditions. 1) The number of granulocytes is 500 / microliter or less. 2) Platelet count is 20,000 / microliter or less. 3) Anemia characterized by hypoplastic bone marrow with a reticulocyte count of less than 1% after conversion and significantly fewer hematopoietic cells. Mild (moderate) aplastic anemia generally shows hypoplasia of the bone marrow, but the reduction of at least two types of cells is not severe. The progression of this type of anemia is potential, with only the first feeling of fatigue due to anemia, and sometimes bleeding. Bleeding is often in the lining of the skin or mucous membrane and is thought to be due to a decrease in platelets. Neutrophils are significantly reduced, but there is little infection. Physical examination of the patient often results in pallor, purpura and punctate bleeding. Patients with aplastic anemia do not have lymphadenoma or splenomegaly, sometimes have fever symptoms, but some do not. Peripheral blood tests show pancytopenia. In general, if immature red blood cells or white blood cells are present, it can be almost determined that it is no longer an aplastic anemia.

  Due to the increasing production pressure of erythrocytes, the erythrocytes are somewhat larger but are effervescent orthochromic. The reticulocyte correction count is very low (may be zero), indicating that no red blood cells are formed. Even if blood coagulation index is normal, bleeding time is long. The iron content of the patient's serum is high, the iron transport protein (transferrin) is normal, and as a result, the transferrin saturation is high. Plasma iron excretion is reduced due to reduced red blood cell production. When a bone marrow puncture is performed, it is found that the bone marrow is usually dry. Biopsy shows severe cell loss or aplastic bone marrow replaced with fat. In some cases, cell hyperplasia may be observed in the initial biopsy, so it is necessary to perform biopsy several times to obtain a correct diagnosis. Myeloid, erythroblastic, pluripotent and megakaryocyte hematopoietic progenitors are all severely depleted. Diagnosis depends on these three conditions: anemia, neutrophil depletion and platelet depletion. X-rays should also be done to exclude bone damage and neoplastic infiltration. Magnetic resonance imaging can be used to clearly determine hypoplastic bone marrow. Aplastic anemia is a disease that is diagnosed by the exclusion method, and other diseases, such as pancytopenia, must be ruled out before making a correct diagnosis.

  Basically, aplastic anemia is the inability to produce various cells. The mechanism and pathogenesis are considered to be in the following four. 1) Defect or lack of hematopoietic stem cells. 2) Abnormal bone marrow microenvironment. 3) Abnormal “regulatory cell”. 4) Decreased hematopoietic function by immune cells.

The pathology of aplastic anemia has not been fully elucidated (Young et al .: The
pathophysiology of acquired aplastic anemia, N. Engl. J. Med. 1997; 336 (19): 1365-1372 and Young et al .: The
treatment of severe acquired aplastic anemia, Blood. 1995; 85 (12): 3367-337), research proved to be an immune disease. For treatment, bone marrow transplantation and immunosuppressive methods using anti-lymphocyte globulin and cyclosporine were adopted (Rosenfeld et al .: Intensive
immunosuppression with antithymocyte globulin and cyclosporine as
treatment for severe aplastic anemia, Blood 1995; 85 (11): 3058-3065, Halperin et al .: Severe acquired aplastic anemia in
children: 11 -year experience with bone marrow transplantation and
immunosuppressive therapy (severe aplastic anemia in children viewed from 11 years of experience in bone marrow transplantation and immunosuppressive therapy), Am. J. Pediatr. Hematol. Oncol. 1989; 11 (3): 304-309). However, immunosuppressive therapy often causes severe side effects. Granulocyte colony-stimulating factor (Kojima et al., Treatment of aplastic anemia in children with recombinant human granulocyte-colony stimulating factor), Blood 1991; 77 (5 ): 937-941, Sonoda et al .: Multilineage response in
aplastic anemia patients following long-term administration of filgrastim, Stem Cells 1993; 11: 543-554), granule Sphere-macrophage colony stimulating factor (Champlin et al .: Treatment of
refactory aplastic anemia with recombinant human granulocyte-macrophage-colony-stimulating factor, Blood 1989; 73 (3): 694-699,
Guinan et al: A phase I / II trial of recombinant granulocyte-macrophage
colony-stimulating factor for children with aplastic anemia, Blood 1990; 76 (6): 1077-1082) and interleukin-3 (I / II stage experiment for childhood aplastic anemia caused by recombinant granulocyte-macrophage colony-stimulating factor)
Ganser et al .: Effect of recombinant human interleukin-3 in patients with normal hematopoiesis and in patients with bone marrow failure, Blood 1990; 76 (4) : 666-676. Nimer et al: A phase I / II study of
interluekin-3 in patients with aplastic anemia and myelodysplasia (I / II stage study for aplastic anemia patients and myelodysplastic syndrome patients), Exp.
Hematol. 1994; 22: 875-880) has limited effects and is only temporary.

  Many patients responded to immunosuppressive therapy, and in patients with aplastic anemia, abnormal amounts of immune molecules were seen. Examples include interleukin-1 made by macrophages, natural killer cells, B lymphocytes and endothelial cells. This interleukin-1 acts on bone marrow stromal cells, induces the generation of unipotent erythroblasts and pluripotent colony-stimulating factors, and restores early progenitor cells and stimulated stem cells to induced myelosuppression Plays an important role in regulating immune response and hematopoietic function. Impaired immune control associated with aplastic anemia is characterized by decreased natural killer cell activity, increased activation-suppressing T cell count, and abnormal occurrence of interleukin-2 and gamma-interferon.

  Natural killer cells are large granular lymphocytes that can lyse these cells when in direct contact with tumor cells and target cells infected with the virus. Natural killer cells can also produce gamma-interferon and interleukin-2, which also induces colony stimulating effects. Under certain conditions, these cells can suppress the formation of myeloid and erythroid colonies. For example, when there are no exogenous growth factors in the culture, natural killer cells generally make cytokines and support hematopoiesis. However, natural killer cells suppress hematopoiesis when optimal conditions are met. The natural killer cell activity returns to normal after the hematopoietic function of the patient with aplastic anemia is restored.

  Gamma-interferon is produced by activated lymphocytes and functions to suppress hematopoiesis. In patients with aplastic anemia, overproduction of gamma-interferon is seen, but the level is reduced with immune control. Interferon moves to progenitor cells directly or indirectly through accessory immune system cells, and has a function of strongly suppressing the formation of hematopoietic colonies.

  Another cytokine overproduced in patients with aplastic anemia is tumor necrosis factor-alpha, which functions to suppress colony growth of normal hematopoietic progenitor cells. The higher the amount of tumor necrosis factor-alpha, the lower the count of platelets, hemoglobin and white blood cells. The synergistic effect of tumor necrosis factor-alpha and gamma-interferon suppresses hematopoietic function.

  In addition to overproduction of gamma-interferon and tumor necrosis factor-alpha in patients with aplastic anemia, the helper-suppressor T cell ratio is also reversed, and the bone marrow has a very high amount of suppressor T cells. Has been generated. These cells can reduce the effect of inhibiting hematopoiesis through the production of cytokines. The proportion of cytotoxic T cells in the patient's bone marrow is also much higher than that of peripheral blood. In view of the decrease in activated lymphocytes after immunosuppressive treatment, aplastic anemia is associated with impaired immune function.

The mechanism of acquired aplastic anemia, particularly benzine-induced aplastic anemia, has not yet been elucidated. However, all aplastic anemia has many pathophysiological and clinical similarities. At present, there are mainly two hypotheses regarding the mechanism of aplastic anemia: direct damage theory and immune participation theory, both of which have been demonstrated by experimental data and clinical studies. Direct damage is thought to be the cause of temporary, reversible bone marrow failure in patients receiving cytotoxic chemotherapy and radiation therapy. Immunized bone marrow failure is very difficult to treat. Benzene-induced aplastic anemia appears to be due to the above two mechanisms. Studies have shown that benzine can damage several biochemical processes in bone marrow cells, particularly bone marrow stromal macrophages, and inhibit interleukin-1 production (Niculescu et al .: Inhibition of the conversion of pre-interleukins- 1 [alpha] and 1 [beta] to mature
cytokines by p-benzoquinone, a metabolite of benzene, the inhibitory effect of p-, a benzine metabolite, on the conversion of pre-interleukin-1 (alpha) and pre-interleukin-1 (beta) into mature cytokines, Chemico -Biological Interactions; 1995; 98: 211-222, Kalf et al:
p-benzoquinone, a reactive metabolite of benzene, prevents the
processing of pre-interleukins-1 [alpha] and -1 [beta] to active
cytokines by inhibition of the processing enzymes, calpain, and
interluekin-1 [beta] converting enzyme (a kind of benzine metabolite)
Inhibition of the conversion of pre-interleukin-1 (alpha) and pre-interleukin-1 (beta) to activated cytokines by inhibiting p-processing enzymes and interleukin-1 (beta) -converting enzymes), Environmental Health Perspectives; 1996; 104 (suppl. 6): 1251-1256). Interleukin-1 is important for stem cells to grow and differentiate (Bagby, GC: Production of multi lineage growth factors by
hematopoietic stromal cells: an intercellular regulatory network
involving mononuclear phagocytes and interleukin-1 (production of multilineage growth factors by hematopoietic stromal cells: intercellular regulatory network containing mononuclear phagocytes and interleukin-1), Blood Cells 1987; 13: 147-159. Fibbe et al: Human
fibroblasts produce granulocyte-CSF, macrophage-CSF and granulocyte-
macrophage-CSF following stimulation by interleukin-1 and poly (r1) .poly (rC) (human granulocyte-CSF, macrophage-CSF and granulocyte by stimulation with interleukin-1, poly (r1) and poly (rC) -Generation of macrophages-CS), Blood 1988; 72 (3): 860-866). However, there have been no reports on long-term response to treatment with hematopoietic growth factors such as interleukin-1.

Medium Ideal culture media for in vitro cultures should generally contain many necessary nutrients such as inorganic salts, amino acids and vitamins that can be used directly by blood cells. An example of such a medium is RPMI 1640, but other mediums such as serum-free medium AIM-V can also support blood cells, and may be used for culture. In addition to the medium, mammalian serum (for example, fetal bovine serum) may be added to the medium in an amount of 0.1 to 50%, 1 to 40%, 5 to 15%, or the like. RPMI 1640, an improved RPMI in the American Type Culture Collection catalog (30-2001), contains the following ingredients:
1.Inorganic Salts (g / liter) Ca (NO.sub.3) .2.4H.sub.2O 0.10000 MgSO.4 (anhydrous) 0.04884 KCl 0.40000 NaHCO.3 1.50000 NaCl 6.00000 Na.sub. 2HPO.sub.4 (anhydrous) 0.80000 Amino Acids (g / liter) L-Arginine (free base) 0.20000 L-Asparagine.H.sub.2O 0.05682 L-Aspartic Acid 0.02000 L-Cystine.2HCl 0.06520 L-Glutamic Acid 0.02000 L-Glutamine 0.30000
Glycine 0.01000 L-Histidine (free base) 0.01500 Hydroxy-L-Proline 0.02000 L-Isoleucine 0.05000 L-Leucine 0.05000 L-Lysine.HCl 0.04000
L-Methionine 0.01500 L-Phenylalanine 0.01500 L-Proline 0.02000 L-Serine 0.03000 L-Threonine 0.02000 L-Tryptophan 0.00500 L-Tyrosine.2Na.2H.sub.2O 0.02883 L-Valine 0.02000 Vitamins (g / liter) D-Biotin 0.00020 Choline Chloride 0.00300 Folic Acid 0.00100 myo-Inositol 0.03500 Nicotinamide 0.00100 p-Amino Benzoic Acid 0.00100 D-Pantothenic Acid 0.00025 (hemicalcium) Pyridoxine.HCl 0.00100 Riboflavin 0.00020 Thiamine.HCl 0.00100
Vitamin B-12 0.000005 Other (g / liter) D-Glucose 4.50000 Glutathione (
reduced) 0.00100 HEPES 2.38300 Phenol Red, Sodium Salt 0.00500 Sodium
Pyruvate 0.11000
1. Cytokines: One or more types of cytokines can be used when culturing activated blood cells according to the present invention. Cytokines are cell-derived small molecular weight proteins (molecular weight 5-20 kD) that have special effects on cell-cell interactions, cell-cell communication and other cellular activities. Cytokines are divided into interleukins, signaling molecules (tumor necrosis molecules) and interferons. In the present invention, in addition to natural cytokines, for example, recombinant cytokines produced using a nucleic acid expression system, or modified or mutated forms of natural cytokines having the function of natural cytokines can be used. The following describes some of the cytokines that can be used in the examples of the present invention.

  A. Interleukin: Interleukin is a naturally occurring polypeptide that can affect the function of specific cells. Interleukins are secreted by regulatory proteins made by lymphocytes, mononuclear cells and other cells, and are released upon antigenic and non-antigenic stimuli. Currently, a total of 16 types of interleukins have been identified. Interleukins regulate inflammatory and immune responses by regulating the growth, activity and differentiation of lymphocytes and other cells. In the present invention, interleukins having concentrations of 10-50,000 IU / ml, 100-5,000 IU / ml, 100-1,000 IU / ml or other effective concentrations are used. “Effective concentration” as used herein refers to the concentration of interleukin used when activated blood cells can be activated when culturing activated blood cells using the method according to the present invention.

  i. Interleukin-1 (IL-1): IL-1 is a soluble protein secreted by monocytes, macrophages or accessory cells that participate in the activation of T and B lymphocytes (17 kD, 152 amino acids) ), Which acts to enhance the response of these cells to antigens or. In addition to replacing macrophages necessary for T cell activation, IL-1 has biological effects such as affecting a wide range of other cell types. At least two IL-1 genes are now known, and IL-1 alpha and beta forms have been identified. IL-1 is released by mononuclear cells and macrophages early in the immune response and promotes T cell proliferation and protein synthesis. Another effect of IL-1 is to generate fever.

  ii. Interleukin-2 (IL-2): IL-2 is a hormonal substance released from stimulated T lymphocytes that activates and differentiates other T lymphocytes regardless of antigen. There is work. It stimulates the growth of blood cells that have certain disease resistance effects of the immune system and, when secreted by Th1 CD4 cells, also has the effect of stimulating CD8 cytotoxic T lymphocytes. IL-2 itself can also promote CD4 cell proliferation and maturation.

  iii. Interleukin-3 (IL-3): IL-3 is a product of T cells activated by mitogenic factors and acts as a colony stimulating factor for bone marrow stem cells and mast cells. IL-3 is also a kind of hematopoietic colony stimulating factor.

  iv. Interleukin-4 (IL-4): IL-4 is a soluble cytokine factor made by activated T lymphocytes, and has the effect of promoting B cell proliferation and differentiation and promoting antibody production. IL-4 can induce the expression of major histocompatibility complexes belonging to class II and fc receptors on B cells. IL-4 also has an effect on hematopoietic cells such as T lymphocytes, mast cells, granulocytes, megakaryocytes, erythroid progenitor cells, and macrophages.

  v Interleukin-5 (IL-5): IL-5 is a factor that promotes eosinophil differentiation and activation of hematopoietic function. It also acts to induce terminal differentiation of activated B cells into Ig secreting cells.

  vi. Interleukin-6 (IL-6): In addition to stimulating the growth and differentiation of human B cells, IL-6 also acts as a growth promoter. IL-6, which can be generated from various cells (T cells, mononuclear cells, fibroblasts, etc.), is a single-chain cytokine (25 kD) and was originally said to be a pre-B cell growth promoting factor. It was recognized that IL-6 also has the effect of stimulating and proliferating other cells (such as T cells).

  vii. Interleukin-7 (IL-7): IL-7 is a B cell growth-promoting factor and, like interleukin-2, also has a mitogenic effect that promotes activation of mature T cells. . IL-7 is made by bone marrow stromal cells.

  viii. Interleukin-8 (IL-8): IL-8 is a cytokine that activates neutrophils and attracts neutrophils and T lymphocytes. In the presence of inflammatory stimuli, IL-8 is released by mononuclear cells, macrophages, T lymphocytes, fibroblasts, endothelial cells and the like. IL-8 belongs to and is structurally related to platelet factor 4.

  ix. Interleukin-9 (IL-9): IL-9 is a cytokine produced by T cells, especially T cells stimulated by mitogens, and has the function of proliferating erythroid progenitor cells (BFUE) It seems to have an effect of controlling hematopoiesis. Can work together with. The IL-9 receptor belongs to the hematopoietic receptor superfamily. IL-9 can promote the growth of human mast cells, megakaryoblastic leukemia cells and mouse helper T cell clones. IL-9 is a glycoprotein derived from a map of T cells and human chromosome 5.

  x. Interleukin-10 (IL-10): IL-10 is a factor produced by Th2 helper T cells, some B cells, and LPS-activated mononuclear cells. There is an effect of.

  xi. Interleukin-11 (IL-11): IL-11 is a multifaceted cytokine originally isolated from primate bone marrow stromal cells that regulates antigen-specific antibody responses and enhances megakaryocytes. It has effects such as controlling the formation of bone marrow fat. IL-11 can stimulate various stages of T cell-dependent B cell maturation, megakaryocyte formation and bone marrow differentiation.

  xii. Interleukin-12 (IL-12): IL-12 cytokine, composed of 40 kD and 35 kD subunits joined together. These subunits were originally recognized as capable of inducing impaired effect cells by co-action with interleukin-2, which does not have the ideal concentration. IL-12 is released by macrophages in response to infection and has the effect of causing cell-mediated immunity. In particular, IL-12 is thought to be an initiator of cell-mediated immunity because it can promote the maturation of Th1 CD4 cells, the reaction of specific cytotoxic T lymphocytes and the activation of NK cells. IL-12 also has the effect of enhancing the cytolytic properties of NK cells, inducing the production of interferon, and stimulating the proliferation of activated T cells and NK cells. IL-12 is produced by human B cells (NC 37).

  xiii. Interleukin-13 (IL-13): IL-13 is a T lymphocyte-derived cytokine that can induce immunoglobulin growth, isotype conversion, and production of immunoglobulin by immature B lymphocytes. IL-13 produced by activated T cells can suppress the production of pro-inflammatory cytokines such as TNF, IL-1 and IL-8, in addition to inhibiting the production of IL-6 by mononuclear cells. The gene for IL-13 is located on human chromosome 5q in the gene population that includes the IL-4 gene.

  xiv. Interleukin-14 (IL-14): IL-14 is a type of cytokine that induces B cell proliferation, suppresses secretion of immunoglobulin, and selectively selects some B cell subpopulations. Has the effect of expanding.

  xv. Interleukin-15 (IL-15): IL-15 is a cytokine that stimulates the proliferation of T lymphocytes and can share the biological activity of IL-2, and also induces the proliferation and differentiation of B lymphocytes is there.

  xvi. Interleukin-16 (IL-16): IL-16 is a cytokine produced by activated T lymphocytes that stimulates migration of CD-4 positive lymphocytes and mononuclear cells.

  B. Lymphokines: Lymphokines are substances that are produced by leukocytes and act on other cells, such as interleukins, interferon alpha, lymphotoxin (tumor necrosis factor alpha), and granulocyte mononuclear cell colony stimulating factor (GM-CSF). is there.

  i. Interferon (IFN): IFN is a type of glycoprotein produced by human cells, and is generally said to have the action of suppressing the growth of viruses and protecting the virus from the human body. In the present invention, the IFN concentration may be substantially the same as the interleukin concentration or may be an effective concentration. The “effective concentration” mentioned here is the concentration of interferon used to realize the activation of blood cells when the blood cells are activated by the method according to the present invention. By the way, IFN alpha and IFN gamma are produced by leukocytes and fibroblasts, respectively, when a viral infection occurs.

  1. Interferon gamma is an interferon produced by T lymphocytes in response to stimulation of specific antigens or mitogens.

  2. Interferon alpha can be divided into several subtypes, both of which are produced by white blood cells when virally infected or stimulated by double-stranded RNA. IFN-alpha-2A and -2B are proteins produced by recombinant DNA technology and are used as antitumor agents. Interferon alpha is a type I interferon produced when peripheral blood leukocytes or cells are stimulated by live viruses, inactivated viruses, double-stranded RNA or bacterial products. Interferon alfa is a major interferon produced by virus-induced leukocyte cultures, and has the effect of activating natural killer cells in addition to its remarkable antiviral properties.

  3. Interferon alpha-2a is a type I interferon with 165 amino acid residues and a lysine at position 23. This protein is produced by DNA recombination technology, is similar to interferon secreted by leukocytes, and is widely used as an antiviral or antitumor agent.

  4. Interferon alpha-2b is a type I interferon with 165 amino acid residues and a lysine at position 23. This protein is produced by DNA recombination technology, is similar to interferon secreted by leukocytes, and is widely used as an antiviral or antitumor agent.

  5. Interferon Better is a type I interferon, a cytokine produced when fibroblasts receive the same stimulus as interferon alfa (by live virus, inactivated virus, or double-stranded RND). Viral, antiproliferative and immunomodulatory.

  6. Interferon-b2 (interleukin-6) is a cytokine that can stimulate the growth and differentiation of human B cells and is a growth promoting factor for hybrid and plasmacytomas. It is produced by many types of cells such as T cells, mononuclear cells, fibroblasts. INF-b2 is a single-chain, 25 kD cytokine, originally said to be a pre-B cell promoter, but recently it has been recognized that it can stimulate the proliferation of other cells such as T cells. INF-b2 can induce acute phase proteins and can also act on the mouse bone marrow as a colony stimulating factor.

  7. Interferon gamma is produced when T cells are stimulated by specific antigens or mitogens.

  ii. Tumor necrosis factor (TNF) is a type of tumor suppressor that appears in the blood when bacterial lipopolysaccharide is present.

  TNF preferentially kills tumor cells in and out of the body, can induce necrosis of certain tumors transplanted into mice, and can suppress experimental transfer. Human TNF alpha is a protein with 157 amino acids and has a wide range of pro-inflammatory properties. In the present invention, the concentration of TNF may be substantially the same as the concentration of interleukin or may be an effective concentration. The “effective concentration” mentioned here is the concentration of TNF used to realize the activation of blood cells when the blood cells are activated by the method according to the present invention.

  C. Cell Stimulating Factor: When activating blood cells for the treatment of aplastic anemia by the method of the present invention, a better effect can be obtained by using one or more types of cell stimulating factors. Such cell stimulating factors include granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor and macrophage colony stimulating factor. In the present invention, cell stimulating factors having a concentration of 10 to 50,000 IU / ml, 10 to 10,000 IU / ml, 10 to 1,000 IU / ml or other effective concentrations are used. The “effective concentration” here refers to the concentration of the cell stimulating factor used when the activated blood cells can be activated when the activated blood cells are cultured using the method according to the present invention.

  1. Granulocyte colony stimulating factor (G-CSF): G-CSF is a glycoprotein synthesized by many types of cells and is involved in the growth and differentiation of hematopoietic stem cells. Furthermore, this type of factor can also stimulate the terminal cell functionality of stem cells.

2. Granulocyte-macrophage colony stimulating factor (GM-CSF):
GM-CSF is a 23 kD, acidic glycoprotein with an internal disulfide bond and is produced by mesenchymal cells in the hematopoietic environment and surrounding inflammation as a response to inflammatory mediators. GM-CSF stimulates bone marrow cells and promotes the formation of neutrophilic granulocytes, macrophages and mixed colonies of granulocytes and macrophages, as well as stimulating fetal liver progenitor cells and promoting the formation of eosinophil colonies it can.

  3. Macrophage colony-stimulating factor (M-CSF): M-CSF is a cytokine synthesized by mesenchymal cells that stimulates pluripotent stem cells of the bone marrow, induces differentiation, and mononuclear cells (mononuclear phagocytes) ). It also acts to stimulate the survival, proliferation and differentiation of mononuclear macrophage hematopoietic cells. M-CSF is a dimer with a molecular weight of 70 kD and a disulfide bond, and binds to a kind of high-affinity receptor. This high affinity receptor is the same as the product of c-fms.

  2. Ionophores: Ionophores are calcium or other cation-specific reagents that can pass through lipid bilayers and fat-soluble layers. Ionophores can be divided into two types: carriers and channel formers. The carrier (eg, valinomycin) forms a cage-like structure around specific ions and can freely diffuse from the hydrophobic region of the double layer. Channel formers (eg, gramicidin) form aqueous pores through the bilayer so that ions can diffuse out of the aqueous pores. In the present invention, in addition to the ionophore, A23187 (calcimycin), ionomycin, geldanamycin, monensin (Na salt), nystatin, polymyxin B sulfate and rapamycin are also included. A carrier such as A23187 appears to act to accumulate calcium cations in response to changes in pH gradient. A23187 has a dissociated carboxylic acid population and can promote neutralization exchange by transposing protons to other cations across the membrane (Hyono et al .: BBA 389, 34-46 (1985). Kolber and Haynes, Biophysics Journal 36, 369-391 (1981), Hunt and Jones, Biosci. Rep., 2, 921-928 (1982)). Since the two molecules of A23187 exist in the form of a carboxylate anion, after carrying and releasing the divalent cation, protons or their equivalents can be brought back into the membrane. If ionophores are added, their concentrations should be 1-10,000 ng / ml, 1-1000 ng / ml, 10-500 ng / ml or other effective concentrations. “Effective concentration” is the concentration of ionophore required to activate (must not be overactivated) blood cells by the method according to the present invention. If the concentration of the active agent is too high, the effect is not good for the treatment method of the present invention.

Use, packaging and distribution Administration of activated cells to a patient can significantly improve the patient's clinical indicators. For example, when the activated cells are given to a patient, it can be seen that the white blood cell count, red blood cell count, hemoglobin level, and platelet count in the patient blood all increase. In general, the patient's blood can be restored to normal levels by continuing treatment according to the present invention. In an embodiment of the present invention, after four treatments, the patient's white blood cell count, red blood cell count, and hemoglobin count all increase by at least 20% and may increase to 35% and 50%. In other examples, the platelet count may have increased 25%, 50%, or even 100%. Of course, there is also an improvement in the blood index within the above range, and since it becomes a part of the present disclosure from the viewpoint of common sense, it is not a luxury here.

  In one embodiment of the present invention, an active compound (eg, one or more types of cytokines and / or one or more types of ionophores) is mixed with an appropriate cell culture medium or a portion thereof for distribution. . In other examples, one or more activating compounds were packaged with the cell culture medium or a portion thereof for convenient transportation. Similarly, for convenience of transportation, one or more kinds of cytokines and one or more kinds of ionophores may be combined and packaged as necessary. In this case, cytokines and ionophores may be mixed or separately packaged. In all of the above examples, the medium or activation compound can be combined with other medium components or activation compounds to form a suitable medium that can activate the cells under activation conditions. Further, in all of the above-described embodiments, in addition to the above components, instructions regarding a method for completing a cell culture medium or a method for cell culture can be attached.

  Cell culture may be performed in a facility for treating a patient or in a remote place. In either case, the harvested activated cells must be administered to the patient as soon as possible while the harvested activated cells are alive. The cells may be stored under conditions that ensure cell survival (for example, at a temperature of liquid nitrogen). Cells are prepared using known methods (eg, making a suspension with buffered saline) at an appropriate time prior to administration to the patient. A known carrier may be used when delivering the cells.

Example 1
Treatment of aplastic anemia
I. Eight patients, each with 1 to 6 years of occupational benzine exposure history. Treatment with the therapy according to the present invention was performed with the consent of the patient. The patient consists of one male and seven females, aged 24 to 41 years. All patients have symptoms of weakness, dizziness, fainting, and high heart rate. Four of them were hospitalized and treated because of bleeding symptoms. Blood or platelets were transfused to hospitalized patients. The remaining four patients were treated for 4 months, 6 months, and 15 months, respectively, because they had chronic symptoms. To confirm hematopoietic function, bone marrow tissue sections and bone marrow puncture samples were taken for all patients. Test results showed that all patients had toxic levels of benzine in their blood and bone marrow.

II. Purification of peripheral blood mononuclear cells and cell culture Blood samples (40-50 ml) of patients were collected, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Hypaque density gradient centrifugation. Separated PBMCs are placed in RPMI1640 (2 × 10 ⁶ cells / ml) containing 10% fetal bovine serum in an appropriate amount (determined by cell concentration), and interleukin-2 (IL-2) 500 IU / ml (Chiron, Emeryville , Calif.), Granulocyte-macrophage colony stimulating factor (GM-CSF) 200 IU / ml (Immunex, Seattle, Wash.) And calcium ionophore A23187 100 ng / ml (Sigma, St. Louis, Mo.) The cells were cultured for 48 hours. After completion of the culture, the cells attached to the plastic surface of the culture vessel were scraped off and collected together with the cells not attached. In order to obtain cells, centrifugal precipitation was performed to make the cell mass into small spheres. Each patient had a different cell yield, but both were washed twice with saline and given to the patient. The washed cells were resuspended in 5-10 ml (determined by the amount of cells) saline and diluted with 50 ml saline prior to administration to the patient.

Treatment One patient (HC) had a very low blood count and had bleeding and infection, so in the first three treatments all activated allogeneic PBMCs were given to this patient. For other patients, activated PBMCs were dissolved in 50 ml saline and injected intravenously. Treatment continued once a week for a total of three weeks. The amount of cells administered to a particular patient is determined by the amount of cells obtained from the patient's blood sample.

III. Results Hematology indices For each patient, pre- and post-treatment blood indices (white blood cell count, red blood cell count, hemoglobin level and platelet count) were recorded (Table 1). These data indicated that the treatment was effective in effectively increasing the patient's peripheral blood cells. Six patients improved more than one item and two patients improved platelet count. Many patients' blood counts improved after receiving the second treatment, and the effect of improvement increased gradually during continuous administration of activated blood cells. At the end of four treatments, of the eight patients, seven patients had their blood indicators restored to normal or near normal. Through this treatment, all patients' blood counts improved, but the degree of improvement was not the same for each patient. For example, some patients showed only a slight improvement in red blood cells, but the platelet count was greatly improved. In any case, all patients' platelet counts increased significantly.

  Unlike other patients, Mr. HC's symptoms were particularly severe and accompanied by bleeding. Since the amount of activated cells obtained from HC's peripheral blood sample was small, the same type of PBMC was administered to stimulate HC's hematopoietic function. Using this PBMC, HC's blood count began to improve after completing the third treatment. After that, I continued treatment using HC's own PBMC. At the end of the six treatments, HC's blood index did not return to normal, but continued improvement was seen anyway.

  Patients who received this immunotherapy were not seen to be mild or moderately inappropriate. Five of them didn't feel such inadequacy at all. Three patients had chills, fever (37-39 ° C), headache, nausea, loss of appetite, etc. after receiving cell infusions, all of which were temporary symptoms and given aspirin It disappeared completely after 2 days.

Bone marrow hematopoietic function Prior to the first treatment and two weeks after the final treatment was completed, patient bone marrow tissue sections and bone marrow puncture samples were taken. As shown in FIG. 1, there was severe damage to the bone marrow of the three patients before treatment, but significant histological improvement was seen after treatment. In the case of patient HC, however, there was no relationship between bone marrow improvement and peripheral blood count improvement.

Prior to transfusion treatment, four of the eight patients were severely associated with bleeding. For these patients, it was determined that it was necessary to infuse whole blood or platelets periodically before and during treatment, but the bleeding stopped when the fourth treatment was completed, so whole blood Or platelet transfusion was stopped.

Period The effect of this cell therapy is not considered to be temporary. This is because all patients' blood indicators remained stable or continued to improve even after treatment was completed. For some female patients, blood counts were not stable during the menstrual period, but no recurrence was seen in all patients. In the case of patient LC, the blood index was stable for two months after the final treatment was completed (Figure 2).

Discussion From the results of this study, it can be said that activated PBMC can be effectively treated for patients with aplastic anemia.

  Although some patients' bone marrow tissues recovered to normal, peripheral blood index was not normal. This is thought to be due to a time gap between bone marrow tissue recovery and peripheral blood recovery. Although continuous observations were made on these patients, the blood index of any patient showed improvement. It took several weeks or months, depending on the patient's time to recover to normal.

  From the data of this study, the improvement in platelets was particularly noticeable compared to other components of blood. This is considered that the production cycle of platelets is shorter than the production cycle of other blood components. In addition to the above four blood components, the neutrophil count, granulocyte count, and reticulocyte count were simultaneously improved, although not listed in the data. Although platelets are blood components that are easily affected by benzine exposure, the introduction of this treatment method and its short generation cycle have contributed to the improvement.

Acquired aplastic anemia is a difficult disease to treat. Until now, there was only a treatment method for bone marrow transplantation for such patients, but this disease (immunosuppressive therapy) can be effectively treated by introducing this treatment method (immunosuppressive therapy). Bone marrow transplantation is also a very effective treatment, but tumors (Socie et al., Malignant tumors occurring
after treatment of aplastic anemia, N. Eng. J. Med. 1993; 329 (16): 1152-1157), (Ferrara et al., Graft-versus-host disease, N. Engl. J. Med. 1991 (324 ); 324: 667-674). Other than that, many patients were unable to receive bone marrow transplants due to high surgical costs and the lack of appropriate donors. Thus, there has been a great demand for treatments that are simpler, more effective and have fewer side effects than can treat aplastic anemia. By introducing the method according to the present invention, aplastic anemia can be treated while maximizing side effects. The present cell therapy (immunotherapy) can also be applied to the treatment of other anemias and bone marrow disorders. Such diseases include, for example, diseases that appear in HIV-infected patients receiving cocktail chemotherapy, bone marrow failure, hereditary aplastic anemia, and idiopathic thrombocytopenic purpura in cancer patients receiving chemotherapy or radiation therapy. Diseases are listed.

  I do not want to limit the mechanism of this immunotherapy to any particular theory, but at this stage, the good therapeutic effect of this immunotherapy is thought to be due to several causes. 1) Activated cells can simultaneously secrete many types of effect factors, some of which have not been elucidated. The synergistic effect of these factors led to the therapeutic effect of this therapy. 2) Activated immune cells produced hematopoietic factors that were not yet known, and brought the effects of this therapy (at least some effects). 3) Immune cells have traveled to the bone marrow, and it is thought that cytokines were given to hematopoietic stem cells and other progenitor cells at a short distance. Also, these activated immune cells stayed for a period of time near the bone marrow, resulting in an improvement in the bone marrow microenvironment. 4) Contact between immune cells and hematopoietic cells may play an important role in the growth and differentiation of hematopoietic cells. 5) Even a small amount of activated immune cells can prevent adverse effects on the hematopoietic function of the immune system. The amount of PCMB obtained from 10 to 100 ml of blood was very small, but this small amount of PCMB had a great influence on bone marrow tissue structure and hematopoietic function.

From the results of previous studies, the effect of the method of the present invention (administration of activated blood cells to a patient) was not considered. There was only one exception. Young et al. (Note 2) administered growth-promoting factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor) to patients and influenced the patient's neutrophil count. This study showed that administration of granulocyte colony-stimulating factor significantly increased both neutrophil and platelet counts. Rather than administering interleukin-3 or a combination of interleukin-3 and granulocyte-macrophage stimulating factor, it was better to simply administer the growth-promoting factor. Liu et al. Found that patients with activated peripheral blood mononuclear cells and interleukin-2 “emphasized” anemia in the patient (Liu et al: Cellular interactions in hemopoiesis, Blood Cells 1987; 13: 101-110 Ettinghausen et al .: Hematologic effects if immunotherapy with lymphokine-activated killer cells and recombinant interleukin-2 in
cancer patients, Blood 1987; 69 (6): 1654-1660).

  The present invention also includes manufacturing matters. Package one or more types of cytokines and ionophores in separate containers, use the containers for culturing activated cells, attach levels, instructions, etc. A culture medium suitable for cultivating cultured cells can be attached.

Example 2
Treatment of Thrombocytopenia This example describes treatment for a female patient (age 1 year 5 months) with idiopathic thrombocytopenic purpura. The patient was diagnosed with idiopathic thrombocytopenic purpura at 9 months.

  The patient initially received routine treatment, such as intravenous corticosteroids and immunoglobulins. Despite the response, patients became more dependent on corticosteroid therapy. In order to maintain platelet levels, patients were constantly receiving corticosteroids and their doses were increasing.

  Thereafter, the patient received treatment with the activated cells. The treatment method was almost the same as in Use Example 1, but the volume of blood sample was 20 ml instead of 40-50 ml. Treatment is once a week and treatment period is 9 weeks. In vitro cultured activated cells were measured on day 1, day 8, day 15, day 22, day 29, day 35, day 42, day 49, day 56 and day 56. Administered to the eye by eye. During the period of immunotherapy with activated cells, conventional therapy such as corticosteroids was stopped. As a result, patient platelets gradually recovered to normal levels (Figure 2). On day 49, the patient had a pulmonary infection and platelet levels dropped significantly, but platelet levels returned to normal as infection recovered.

  Publications, patents, patent applications and other documents cited herein are all united by reference to each other. If any doubt arises, it shall conform to this specification.

  Since the present invention may be modified on the assumption that the gist of the present invention is not impaired, the scope of the present invention is not limited to the scope of the above embodiments. The scope of the invention is determined by the appended claims or their equivalents.

FIG. 1 is a low magnification view of H & E stained bone marrow tissue sections of three patients who received this treatment. A, C and E are bone marrow before treatment of the patient. The hollowed, damaged bone marrow exhibits severe aplastic anemia. B, D, and F are bone marrow after treatment, and as shown in the figure, it can be seen that both the distribution and density of bone marrow cells are improved. FIG. 2 shows the platelet count of a child patient with platelet aplastic anemia who was treated.

Claims (31)

A method for culturing blood cells, comprising a method for culturing blood cells in the presence of cytokines and ionophores. The method according to claim 1, wherein the blood cells are cultured under conditions where an effective amount of cytokine and ionophore is present. The method according to claim 1, wherein the blood cells are cultured in a medium containing mammalian serum. The method according to claim 1, wherein the culture period of the blood cells is approximately 10 to 90 hours. The method according to claim 1, wherein the blood cells are cultured in a temperature range of 30 to 42 ° C. The method according to claim 1, wherein the blood cells are cultured under conditions in which interleukin-2 and granulocyte-macrophage colony-stimulating factor are present. The method according to claim 1, wherein blood cells of the same type as the blood cells of the patient are used. The method according to claim 1, characterized in that blood cells from a donor that can be accepted from the viewpoint of immunity are used. 2. The method of claim 1, wherein the patient's own blood cells are used. The method according to claim 1, wherein the blood cells are cultured under conditions where A23187 is present. The method according to claim 1, wherein blood cells or a part thereof used for the culture are separated from the serum in advance. 2. The method according to claim 1, wherein blood cells used for the culture or a part thereof are separated from serum in advance by centrifugation. A treatment method characterized by increasing the concentration of a blood component of a patient by giving blood cells cultured in vitro to an anemia patient. 14. The method of claim 13, wherein the patient is provided with a therapeutically effective amount of blood cells. The method according to claim 13, wherein the blood cells are cultured under conditions where cytokines and ionophores are present. 14. The method according to claim 13, wherein the blood cells are cultured under conditions where a cytokine and an ionophore containing A23187 are present. 14. The method according to claim 13, wherein the blood cells are cultured under conditions where cytokines containing interleukin-2 are present. 14. The method according to claim 13, wherein the blood cells are cultured under conditions where a cytokine containing a macrophage colony stimulating factor is present. 14. The method of claim 13, wherein the patient is provided with a therapeutically effective amount of blood cells. 14. The method of claim 13, wherein the patient's own blood cells are used. 14. The method according to claim 13, wherein blood cells of the same type as the patient's blood cells are used. 14. The method of claim 13, wherein the blood cells are cultured using mammalian serum. 14. The method according to claim 13, wherein blood cells are cultured using fetal calf serum. 14. The method according to claim 13, wherein blood cells from a donor judged to be immunologically acceptable are used. An effective amount of activated blood cells that can treat anemia by injection. 26. The blood cell according to claim 25, which is cultured under conditions in which a cytokine and an ionophore are present. 27. The blood cell according to claim 26, which is cultured under conditions where cytokines and ionophores having effective concentrations are present. 26. The therapeutically effective amount of blood cells according to claim 25, characterized in that the peripheral blood cell count of the patient is increased by giving to the patient. The blood cell according to claim 25, which is cultured under conditions in which interleukin and ionophore are present. The blood cell according to claim 25, which is cultured under conditions in which interleukin-2, macrophage colony-stimulating factor and calcium ionophore are present. The blood cell according to claim 25, which is cultured under conditions in which interleukin-2, granulocyte-macrophage colony stimulating factor and A23187 are present.

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